Site-specific identification and quantitation of endogenous SUMO modifications under native conditions

Ryan J. Lumpkin, Hongbo Gu, Yiying Zhu, Marilyn Leonard, Alla S. Ahmad, Karl R. Clauser, Jesse G. Meyer, Eric J. Bennett () and Elizabeth A. Komives ()
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Ryan J. Lumpkin: Department of Chemistry and Biochemistry, University of California, San Diego
Hongbo Gu: Proteomic Service Group Cell Signaling Technology
Yiying Zhu: Proteomic Service Group Cell Signaling Technology
Marilyn Leonard: University of California, San Diego
Alla S. Ahmad: Department of Chemistry and Biochemistry, University of California, San Diego
Karl R. Clauser: Broad Institute of MIT and Harvard
Jesse G. Meyer: Department of Chemistry and Biochemistry, University of California, San Diego
Eric J. Bennett: University of California, San Diego
Elizabeth A. Komives: Department of Chemistry and Biochemistry, University of California, San Diego

Nature Communications, 2017, vol. 8, issue 1, 1-11

Abstract: Abstract Small ubiquitin-like modifier (SUMO) modification regulates numerous cellular processes. Unlike ubiquitin, detection of endogenous SUMOylated proteins is limited by the lack of naturally occurring protease sites in the C-terminal tail of SUMO proteins. Proteome-wide detection of SUMOylation sites on target proteins typically requires ectopic expression of mutant SUMOs with introduced tryptic sites. Here, we report a method for proteome-wide, site-level detection of endogenous SUMOylation that uses α-lytic protease, WaLP. WaLP digestion of SUMOylated proteins generates peptides containing SUMO-remnant diglycyl-lysine (KGG) at the site of SUMO modification. Using previously developed immuno-affinity isolation of KGG-containing peptides followed by mass spectrometry, we identified 1209 unique endogenous SUMO modification sites. We also demonstrate the impact of proteasome inhibition on ubiquitin and SUMO-modified proteomes using parallel quantitation of ubiquitylated and SUMOylated peptides. This methodological advancement enables determination of endogenous SUMOylated proteins under completely native conditions.

Date: 2017
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DOI: 10.1038/s41467-017-01271-3

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